Introduction to light microscopy - ZMB · PDF file Statistical microscopy – Imaging...

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Transcript of Introduction to light microscopy - ZMB · PDF file Statistical microscopy – Imaging...

  • Center for Microscopy and Image Anaylsis

    Introduction to light

    microscopy

    Basic concepts of imaging with light

    Urs Ziegler

    ziegler@zmb.uzh.ch

  • Light interacting with

    matter

    Absorbtion

    Refraction

    Diffraction

    Scattering

  • Light interacting with

    matter

    Absorbtion

    Refraction

    Diffraction

    Scattering

  • Light interacting with

    matter

    Light emitted from fluorochromes

    How is an image formed?

    Why are there limits in resolution?

  • Introduction to light

    microscopy

    Basic concepts of light microscopical imaging

    - General setup of microscopes

    - Image formation by diffraction and

    interference

    - Resolution limits

    - Light emission from molecules and

    fluorescent imaging

    Methods and techniques in microscopy

    - Summary of microscopical techniques

  • Fundamental Setup of

    Light Microscopes

  • Fundamentals of light

    microscopy

    The objective forms a magnified

    image of the object near (or in) the

    eyepiece

    The intermediate image is

    examined by the eyepiece and

    eye forming a real image on the

    retina

    F eyepiece

    Primary image Eyepiece

    F objective

    Object Objective

    Virtual image seen by eye

  • Fundamentals of light

    microscopy

    Compound microscope:

    Microscope composed of an

    objective and an additional lens

    (eyepiece, occular, tube lens)

    Magnification:

    What is the maximal

    magnification?

    Is there a limit in useful

    magnification?

    Mfinal = Mobjecive x Moccular

    => Why is there a limit in resolution?

  • Properties of light

  • Diffraction and

    interference

    Diffraction and interference: the

    key principles to understand how

    a microscope forms an image

    Diffraction and interference are

    phenomena of wave optics

    Light carries information about the

    object and creates the image in

    the focal plane of a lens

    Object

    Diffraction of light after passing

    object (grating)

  • Diffraction at an aperture

    or substrate

    Disturbance of the electric field of

    a planar wavefront by diffraction

    upon passage through an

    aperture

    A mixture of particles diffracts an

    incident planar wavefront

    inversely proportional to the size

    of particles

  • Image formation in the

    microscope

    Ernst Abbe (1840 – 1905)

    developed the theory for image

    formation in the light microscope

    Diffracted light from a periodic

    specimen produces a diffraction

    pattern of the object in the back

    focal plane

  • Image formation in the

    microscope

  • Image formation in the

    microscope

    Diffracted light from a periodic

    specimen produces a diffraction

    pattern of the object in the back

    focal plane

    Not interacting incident light is

    transmitted undeviated and

    produce the evenly illuminated

    image plane

    Diffraction spots in the back focal

    plane correspond to constructive

    interference of waves differing in

    1, 2, …. wavelengths.

    Image formation in the image

    plane is by interference of

    undeviated and deviated waves

  • Diffraction patterns in the

    back focal plane

    Generation of an image by

    interference requires collection of

    two adjacent orders of diffracted

    light!

    a,b: no image is formed

    c: image is formed

    d: image with high definition due

    to multiple diffracted orders

    collected

  • Diffraction image of a point

    source of light

    The image of a self-luminous point

    in a microscope is a pattern

    created by interference in the

    image plane

    The pattern is a central bright spot

    surrounded by a series of rings

    The central spot contains ≈ 84%

    of light

    The image is called: Airy disk

    (after Sir George Airy (1801 –

    1892))

    http://www.cambridgeincolour.com

  • Spatial resolution in x,y

    and z

    Implications:

    Objects smaller than the

    resolution limit of the chosen

    objective will always be 1Airy disk

    Objects larger than the resolution

    limit of the chosen objective will

    always be the size of the object

    convolved with the optical transfer

    function

    Note: the optical transfer function

    is a function describing how the

    imaging is occurring in the

    microscope

    1 µm

    Crossection

    focal plane

    0.1 µm bead

    Reality

    Theory

    1 µm

    Crossection

  • Resolution and aperture

    angle

    Concept:

    Object is approximated with self

    luminous points

    Image of each individual point is

    not influenced by any other points

  • Resolution and aperture

    angle

    The image of a self luminous point

    is an airy disk

    The self luminous point generates

    a spherical wavefront

    Ideally the path length between

    object and corresponding

    conjugate image is preserved

    Smaller apertures increase the

    size of airy disks

  • Resolution and aperture

    angle

    The objective aperture must

    capture light from a wide angle for

    maximum resolution (diffracted or

    emitted light)

    NA = n sin α

    α: half angle of the cone of

    specimen light accepted by the

    objective

    n: refractive index of medium

    between lens and specimen

    α α

  • Objective with high

    aperture (NA 1.25)

    Objective with low

    aperture (NA 0.3)

    Resolution and aperture

    angle

    Aperture of objective determines

    the resolution, not the

    magnification!

  • Resolution and Rayleigh

    criterion

    Resolving power of microscope:

    𝑑 = 0.61 × λ

    𝑁𝐴

    Concept: an image of an

    extended object consists of a

    pattern of overlapping diffraction

    spots

    Resolution: the larger the NA of

    the objective, the smaller the

    diffraction spots (airy disks).

    a) Single diffraction pattern

    b) Two Airy disks with maximum of one overlapping

    first minimum of the other

    objects just resolved

    c) Two Airy disks with maximum of one overlapping

    the second minimum

    objects well resolved

  • Resolution and size of Airy

    disk

    Concept: an image of an

    extended object consists of a

    pattern of overlapping diffraction

    spots

    Resolution: the larger the NA of

    the objective, the smaller the

    diffraction spots (airy disks).

    Note: this theme of diffraction

    limited spots and their separation

    in space and time will again be

    used and taken up in

    superresolution microscopy.

  • Spatial resolution in x,y

    and z

    Objects are (always) 3

    dimensional

    The resulting ‘image’ will also be a

    3D ‘image’ in the image space

    Again:

    an image of an extended object

    consists of a pattern of

    overlapping diffraction spots

    1 µm

    Crossection

    focal plane

    0.1 µm bead

    Reality

    Theory

  • Out of focus

    Resolution and size of Airy

    disk

    Objects are (always) 3

    dimensional

    The resulting ‘image’ will also be a

    3D ‘image’ in the image space

    Again:

    an image of an extended object

    consists of a pattern of

    overlapping diffraction spots

    Take home:

    In widefield microscopy the out of

    focus information is increasing the

    background and results in low

    contrast images

    In focus

  • Resolution and size of Airy

    disk

    Objects are (always) 3

    dimensional

    The resulting ‘image’ will also be a

    3D ‘image’ in the image space

    Again:

    an image of an extended object

    consists of a pattern of

    overlapping diffraction spots

    Take home:

    In widefield microscopy the out of

    focus information is increasing the

    background and results in low

    contrast images

  • Resolution limits

    𝑑𝑥𝑦 = 0.61 × λ

    𝑁𝐴

    𝑑𝑧 = 𝑛 × λ

    𝑁𝐴2

    These formula are used for the

    calculation of resolution